Hepatotoxicity of new drugs, which is often unpredictable in the preclinical phase of drug discovery, causes failure of 50% of pharmaceuticals that make it to Phase I clinical trials, resulting in ~2-6yr loss in time and ~$15 million in resources per drug. Current in vitro drug screening platforms typically do not consider zonation in hepatocyte metabolism, which is critical for accurate modeling of in vivo function as biotransformation occurs to different extents in different zones, affecting hepatotoxicity. Although many metabolic zonal inducers have been studied, most notably O2, we still lack a systematic understanding of what concentration and concentration gradients of the chemical inducers lead to physiological zonation. Our long-term goal is to create a physiologically relevant, high-throughput system for predictive in vitro drug-screening. Our objective is to develop a systematic understanding of how concentration gradients in chemical agents induce hepatic metabolic zonation and incorporate this understanding into a high-throughput microfluidic screening platform for rapid drug toxicity screening. The central hypothesis is that dynamic zonation of hepatocyte metabolism can be generated using concentration gradients in zonation induction agents (O2, hormones, bile acids, and ethanol) to direct the development of morphologically and metabolically distinct zones within a microfluidic hepatocyte cell culture to elucidate xenobiotic hepatotoxicity. The rationale for this proposal is that current microfabrication technology allows the use of microfluidics to create more physiological liver models that incorporate hepatocyte heterogeneity. Furthermore, such microfluidic platforms can be multiplexed for high- throughput analysis, allowing precisely controlled and efficient drug screening. Aim 1: Design and fabricate a multiplexed microfluidic platform for hepatocyte culture to systematically determine the concentration effects of zonation inducers on cell metabolism. Aim 2: Create a microfluidic platform to assess the effects of concentration gradients of single and multiple zonation induction agents on hepatocyte dynamic metabolism. Aim 3: Create hepatocyte metabolic zonation in the flow-direction via cellular consumption, mimicking liver physiology, and test the hepatotoxicity of various drugs in long-term culture. The successful outcome of these aims will provide novel in vitro microfluidic culture systems capable of reproducing zonal hepatocyte morphology and metabolism. We expect to elucidate the connection between gradients of zonal inducers along the acinus and hepatocyte dynamic metabolic responses, and to correlate metabolic response to drug metabolism and toxicity. The broader impact of the studies will be the development of a predictive, high- throughput in vitro drug-screening platform to replace poorly predictive animal models.